Electrophoresis is a well-known technique for the separation of charged species by utilizing their differences in rate of migration under the influence of an electrical field. The prototype of all modern electrophoretic methods is free, or moving-boundary, electrophoresis. The mobility .mu. in square centimeters per volt-second of a molecule in an electric field is given by the ratio of the velocity of migration v, in centimeters per second, to electric field strength E, in volts per centimeter:.mu.=V/E. For small ions, such as chloride, .mu. is between 4 and 9 .times. 10.sup.-4 cm.sup.2 v.sup.-1 s.sup.-1 (25.degree. C.); for proteins, it is about 0.1 to 1.0 .times. 10.sup.-4 cm.sup.2 v.sup.-1 s.sup.-1 . Protein thus migrates much more slowly in an electrical field than small ions simply because they have a much smaller ratio of charge to mass.
Free electrophoresis has been largely supplanted by various forms of zone electrophoresis in which the aqueous protein solution is immobilized in a solid matrix that provides mechanical rigidity and reduces convection and vibration disturbances. Matrix material that is porous also allows for sieving. This form of zone electrophoresis can separate a protein mixture on the basis of both electric charge and molecular size, thereby providing high resolution.
Capillary zone electrophoresis ("CZE") in small bore capillaries was first demonstrated by Jorgenson and Lukacs, and has proven useful as an efficient method for the separation of certain small solutes. J. Chromatog., 218 (1981), page 209; Anal. Chem., 53 (1981) page 1298. Attractive factors for CZE include the small sample sizes, little or no sample pretreatment, high resolution, automation, and the potential for quantification and recovery of biologically active samples. For example, U.S. Pat. No. 4,675,300, inventors Zare et al., issued June 23, 1987 describes theories and equipment for electrokinetic separation processes employing a laser-excited fluorescence detector. The system described by Zare et al. includes a fused silica capillary with a 75.mu. inner diameter.
CZE can be strongly influenced by electroosmosis, which is the flow of liquid that occurs when an electrical potential is applied to a liquidfilled porous medium. Jorgenson and Lukacs reported that separation of model proteins, such as cytochrome, lysozyme and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was complicated by the adsorption of proteins to the surface of the capillaries. They concluded that adsorption affects electropherograms in two undesirable ways. First, it leads to broad asymmetric "tailed" zones. Second, adsorbed proteins modified the capillary surface, usually decreasing electroosmotic flow significantly which leads to unpredictable migration for all sample zones upon repeated injection. Jorgenson and Lukacs, Science, 222 (1983) page 266.
Electrophoresis in large diameter capillaries may be attended by a hydrodynamic reflow, which causes the zones to become strongly parabolically distorted. A reflow occurs when the resistance to hydrodynamic flow in the electrophoresis tube is relatively low. Reflow becomes more problematic as the inner diameter of the tube increases. Another phenomenon associated with large diameter open tubes is the problem of convection.
It has been reported that coating electrophoresis tubes with a mono-molecular layer of a non-crosslinked polyacrylamide significantly reduces the electroendosmosis effect. Hjerten, S., J. Chromatography, 347 (1985), 191-198. Moreover, the use of anticonvective agents in capillaries reduced the convection problem and allows for the use of higher inner diameter capillaries.
Lauer and McManigill, Anal. Chem., 58 (1986), page 166, have reported that the Coulombic repulsion between proteins and the capillary wall of silica capillaries can overcome adsorption tendencies of the proteins with the capillary wall. They demonstrated separations of model proteins (ranging in molecular weight from 13,000 to 77,000) by varying the solution pH relative to the isoelectric point (pI) of the proteins to change their net charge.
Increasing the selectivity control of capillary electrophoresis has been achieved through the use of anionic micelles from sodium dodecyl sulfate (SDS). This approach has been used to separate bases, nucleosides and nucleotides in a buffer solution with a pH of 7. Since the bases and nucleosides are uncharged at the pH of operation, separation is a result of differential partition within the interior of the micelle; the more hydrophobic the species, the larger the partition coefficient and the larger the retention. Oligonucleotides are negatively charged and can be separated without SDS micelles; however, the time window is narrow and separation of complex mixtures is limited. The combination of low concentrations of divalent metals and SDS micelles leads to a significant enhancement of the time window and good separation of oligonucleotides. The metal ion is electrostatically attracted to the surface of the micelle and differential metal complexation of the oligonucleotides with the surface of micelles leads to separation of complex mixtures. See Cohen, AnaI. Chem., 59 (1987) 1021-27.
Gels as a CZE media are difficult to retain in the capillary tubing. One possible explanation is that an electric double layer forms with the gel structure at the gel-solution interface which causes an appreciable electrostatic flow (EOF) component. Moreover, charged groups within the gel structure may be present. Therefore, between the considerable EOF component and the tendency of the gel to electrophorese due to its own charge, there is a net movement of the gel unless the gel is sufficiently adhered to the surface of the capillary tube.
U.S. Pat. No. 4,810,456, inventors Myerson et al., issued March 7, 1989, describes a method for preparing electrophoretic gel that is substantially free of defects caused by shrinkage that generally accompanies polymerization. The procedure consists of compressing the prepolymer-bearing (e.g., monomer) to a density within a predetermined range and maintaining the density of the substance during polymerization.
U.S. Pat. No. 4,680,201, inventor Hjerten, issued July 14, 1987, describes a method for preparing a thin-wall, narrow-bore capillary tube for free electrophoretic separations by use of a bifunctional compound in which one group reacts specifically with the glass wall and the other with a monomer taking part in a polymerization process. This procedure results in a polymer coating, such as polyacrylamide coating, and is suggested for use in coating other polymers, such as poly(vinyl alcohol) and poly(vinylpyrrolidone). The method purportedly overcomes problems associated with adsorption and electroendosmosis, but this approach introduces a coating phase consisting of material different from the gel medium. Moreover, this method is limited to gel media formed by polymerization such as polyacrylamide which may contain potentially damaging polymerization by-products.